Dna-based molecular switches and uses thereof

Abstract

Disclosed are nucleic acid-based molecular switches that respond to changes in pH. The switches may be used in DNA nanodevices. The switches may also act as sensors for measuring the pH of a sample, including cells, regions thereof, and whole organisms. The switch includes an A-motif that forms at acidic pH. Also disclosed are compositions and methods for measuring the pH of cells or regions thereof, such as vesicles, the nucleus, mitochondrial matrix, or the Golgi lumen.

1 .- 20 . (canceled) 
     
     
         21 . A method for determining a capacity of an agent to effect pH, the method comprising:
 contacting a test sample with a) one or more indicators having at least two poly dA nucleic acid strands comprising a signaling system, wherein the one or more indicators transition between first and second stable conformations in response to a change in pH, and b) the agent, and   detecting the presence, absence, or magnitude of a signal from the signaling system to determine the pH of the sample contacted by the agent   
       whereby a change in the presence, absence, or magnitude of the signal compared to the signal detected in a sample not contacted by the agent indicates the capacity of the agent to effect pH. 
     
     
         22 . The method of  claim 21 , wherein the signaling system comprises an interacting label pair. 
     
     
         23 . The method of  claim 22 , wherein the interacting label pair comprises a fluorophore and quencher pair. 
     
     
         24 . The method of  claim 23 , wherein the fluorophore and the quencher are on separate poly dA nucleic acid strands, such that a difference in a fluorescent signal is detectable upon a change in conformation of the one or more indicators. 
     
     
         25 . The method of  claim 21 , wherein the test sample comprises one or more cells or organelles. 
     
     
         26 . The method of  claim 21 , wherein the one or more indicators further comprise one or more of a fusogenic peptide, a membrane permeabilizing peptide, a subcellular localization signal, or a cell receptor ligand. 
     
     
         27 . The method of  claim 26 , wherein the subcellular localization signal targets the indicator to a region of the cell selected from the group consisting of the cytosol, the nucleus, the endoplasmic reticulum, the mitochondrial matrix, a chloroplast lumen, the medial trans-Golgi cisternae, the lumen of a lysosome, and the lumen of an endosome. 
     
     
         28 . The method of  claim 26 , wherein the subcellular localization signal is selected from the group consisting of a receptor ligand, a nuclear localization signal (NLS), a nuclear export signal (NES), a plasma membrane targeting signal, a histone binding protein, and a nuclear protein. 
     
     
         29 . The method of  claim 26 , wherein the fusogenic peptide is a peptide of a viral protein derived from a virus selected from the group consisting of an influenza virus, a vesicular stomatitis virus, and an alpha virus. 
     
     
         30 . The method of  claim 29 , wherein the fusogenic peptide is a hemagglutinin of an influenza virus or a glycoprotein of a vesicular stomatitis virus. 
     
     
         31 . A kit for determining the pH of a sample, the kit comprising: one or more indicators having at least two poly dA nucleic acid strands comprising a signaling system, wherein the one or more indicators transition between first and second stable conformations in response to a change in pH, and optionally instructions for use. 
     
     
         32 . The kit of  claim 31 , wherein the signaling system comprises an interacting label pair. 
     
     
         33 . The kit of  claim 32 , wherein the interacting label pair comprises a fluorophore and quencher pair. 
     
     
         34 . The kit of  claim 33 , wherein the fluorophore and the quencher are on separate poly dA nucleic acids, such that a difference in a fluorescent signal is detectable upon a change in conformation of the one or more indicators. 
     
     
         35 . The kit of  claim 31 , wherein the one or more indicators further comprise one or more of a fusogenic peptide, a membrane permeabilizing peptide, a subcellular localization signal, or a cell receptor ligand. 
     
     
         36 . The kit of  claim 35 , wherein the subcellular localization signal targets the indicator to a region of the cell selected from the group consisting of the cytosol, the nucleus, the endoplasmic reticulum, the mitochondrial matrix, a chloroplast lumen, the medial trans-Golgi cisternae, the lumen of a lysosome, and the lumen of an endosome. 
     
     
         37 . The kit of  claim 35 , wherein the subcellular localization signal is selected from the group consisting of a receptor ligand, a nuclear localization signal (NLS), a nuclear export signal (NES), a plasma membrane targeting signal, a histone binding protein, and a nuclear protein. 
     
     
         38 . The kit of  claim 35 , wherein the fusogenic peptide is a peptide of a viral protein derived from a virus selected from the group consisting of an influenza virus, a vesicular stomatitis virus, and an alphavirus. 
     
     
         39 . The kit of  claim 38 , wherein the fusogenic peptide is a hemagglutinin of an influenza virus or a glycoprotein of a vesicular stomatitis virus. 
     
     
         40 . A nucleic acid assembly comprising one or more modules comprising an A-motif forming nucleic acid, wherein the A-motif forming nucleic acid-transitions between first and second stable conformations in response to a change in pH in order to join the modules in a nucleic acid assembly, wherein at least one A-motif-forming nucleic acid is selected from the group consisting of 5′-AAAAAAAAAA AAAAAACATG CCGGTATTT AAAGCCTTTC GAAGATATCG TGGTTCGAGA AAAAAAAAAA AAAAA-3′ (SEQ ID NO: 5), 5′-CGAGCTGCAG CAGCTGTTAT TTAGGCTTTA AATACCGGCA TG-3′ (SEQ ID NO: 6), 5′-CTCGAACCAC GATATCTTCG TTATAACAGC TGCTGCAGC TCG-3′ (SEQ ID NO: 7), 5′-ATGCCGGTAT TTAAAGCCT TTCGAAGATA TCGTGGTTCG AG-3′ (SEQ ID NO: 8), and 5′-TTTTTTTTTT TTTTTTCATG CCGGTATTTA AAGCCTTTCG AAGATATCG TGGTTCGAG TTTTTTTTT TTTTTTT-3′ (SEQ ID NO: 9).